A thorough understanding of the mechanism of protein synthesis is valuable because of its importance in cellular metabolism and because gene expression may be controlled in part at the level of translation. The broad goals of the project are to elucidate the molecular and control mechanisms of initiation of protein synthesis in mammalian cells. We shall study in particular the structure and function of the eight initiation factors from rabbit reticulocytes recently purified to near-homogeneity in this laboratory and elsewhere. It is proposed to prepare and purify antibodies to use as specific inhibitors of protein synthesis and as tools for isolating and identifying factors. The kinetic parameters of the interaction of initiation factors with ribosomes and other components of initiation will be studied by fluorescence polarization techniques. The structure of the multi-polypeptide factor, eIF-3, the composition of factor binding sites on ribosomes, and factor-factor interactions will be examined by crosslinking techniques. Construction of a system with purified components for assaying initiation, and the knowledge gained from the experiments on mechanism above, should provide a sound basis for studying at the molecular level those processes which regulate the rate of initiation. Particular attention will be directed to determining how phosphorylation of initiation factors affects initiation rates in reticulocyte and HeLa cells. Explanations will be sought for how initiation of protein synthesis is inhibited following polio virus infection, during the mitotic phase of the cell cycle and when cells are starved of an essential amino acid. It is hoped to integrate our knowledge of the mechanism of initiation of protein synthesis and translational control within the broader context of metabolic control, gene expresson and cellular function.